Diploids of the normally haploid Bacillus subtilis can be produced by polyethylene glycol mediated fusion of protoplasts. Such diploids segregate either one or the other of the parental chromosomes and become haploid again or remain diploid by inactivating one or the other of the parental chromosomes. The latter diploids replicate an inactive chromosome for many generations but do not produce messenger RNA from it. The mechanisms of chromosome inactivation and the ability of such inactivated chromosomes to be transcribed is the focus of study of this proposal. Strains will be constructed in which fusions can be carried out with parental strains bearing chromogenic markers such that the contribution of each parental chromosome can be assayed in situ in the product of the fusion of protoplasts. In addition multiauxotrophic strains will be generated using a deletion insertion mutation such that both parental chromosomes can be followed individually by hybridization. The basic goal of this research is to determine whether non-complementing diploids contain one expressed and one unexpressed chromosome or whether non-expressed chromosomes can be partially transcriptionally active. As a means to approach this end a restriction map of the chromosome will be constructed using restriction endonucleases Not-I and Sfi-I in combination with pulsed-field gradient electrophoresis. The role of cytosine and adenine methylation in the suppression of transcriptional activity from the non-expressed chromosome will be determined. The role of spoOA gene product on the stabilization of non-complementing diploids to segregation will be assayed. The genes involved in the stabilization of non-complementing diploids will be identified and isolated.